Process for preparing distamycin derivatives

ABSTRACT

It is described a process for preparing, in high yields and purity and without the need of carrying out several steps and/or isolating many intermediates which could lead to undesired by-products, a distamycin derivative of formula 
                 
 
wherein R is a bromine or chlorine atom; or a pharmaceutically acceptable salt thereof. The compounds of formula (I) are useful in therapy as antitumor agents.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a process for preparing distamycinderivatives and, more in particular, it relates to a process forpreparing acryloyl-distamycin-guanidino derivatives, known to possess aremarkable antitumor activity.

2. Description of the Related Art

Distamycin A, whose formula is reported below

belongs to the family of the pyrroleamidine antibiotics and it isreported to interact reversibly and selectively with DNA-AT sequences,thus interfering with both replication and transcription. See, for areference, Nature, 203, 1064 (1964); FEBS Letters, 7 (1970) 90; Prog.Nucleic Acids Res. Mol. Biol., 15, 285 (1975).

Several analogues to distamycin are known in the art as antitumoragents.

As an example, the international patent application WO 98/04524 in thename of the Applicant itself, discloses distamycin derivatives, havingvaluable biological properties as antitumor agents, wherein thedistamycin formyl group is replaced by an acryloyl moiety and theamidino group is replaced by several nitrogen-containing ending groups,among which is guanidino.

Specific examples of this class of acryloyl-distamycin-guanidinoderivatives, optionally in the form of pharmaceutically acceptablesalts, for instance as hydrochloride salts, are:

N-(5-{[(5-{[(5-{[(2-{[amino(imino)methyl]amino}ethyl)amino]carbonyl}-1-methyl-1H-pyrrol-3-yl)amino]carbonyl}-1-methyl-1H-pyrrol-3-yl)amino]carbonyl}-1-methyl-1H-pyrrol-3-yl)-4-[(2-bromoacryloyl)amino]-1-methyl-1H-pyrrole-2-carboxamide(internal code PNU 166196); and

N-(5-{[(5-{[(5-{[(2-{[amino(imino)methyl]amino}ethyl)amino]carbonyl}-1-methyl-1H-pyrrol-3-yl)amino]carbonyl}-1-methyl-1H-pyrrol-3-yl)amino]carbonyl}-1-methyl-1H-pyrrol-3-yl)-4-[(2-chloroacryloyl)amino]-1-methyl-1H-pyrrole-2-carboxamide.

These derivatives are prepared according to a chemical processcomprising, essentially, the condensation reaction between a properlyactivated carboxylic acid derivative with a poly-pyrroleamido frameworkbearing the desired nitrogen-containing ending group, for instance theguanidino group.

This latter guanidino intermediate, in its turn, is prepared accordingto a rather troublesome step-by-step procedure which implies,substantially, several acylation reactions of 2-carboxy-4-amino-pyrroleswhich are obtained through reductions of the corresponding nitroderivatives, in a serial manner.

For a general reference to the above process for preparingacryloyl-distamycin derivatives, also includingacryloyl-distamycin-guanidino derivatives see, for instance, theaforementioned WO 98/04524.

BRIEF SUMMARY OF THE INVENTION

In this respect, we have surprisingly found that the saidacryloyl-distamycin-guanidines can be advantageously prepared through achemical process which allows to obtain the desired products in highyields and purity and in a limited number of steps.

Therefore, it is a first object of the present invention a process forpreparing a distamycin derivative of formula

wherein R is a bromine or chlorine atom; or a pharmaceuticallyacceptable salt thereof; which process comprises:

-   -   a) reacting, under basic conditions, 2-amino-ethylguanidine with        a compound of formula    -    wherein X is hydroxy or a suitable leaving group, so as to        obtain a compound of formula    -   b) reducing the nitro-derivative of formula (III) to the        corresponding amino-derivative and, subsequently, reacting the        resultant amino-derivative with the above compound of formula        (II), under basic conditions, so as to obtain a compound of        formula    -   c) reducing the nitro-derivative of formula (IV) to the        corresponding amino-derivative and, subsequently, reacting in        the presence of a suitable condensing agent and under basic        conditions the resultant amino-derivative with a compound of        formula    -    wherein R is a bromine or chlorine atom; so as to obtain the        compound of formula (I) and, optionally, converting it into a        pharmaceutically acceptable salt thereof.

The process object of the present invention allows to obtain thecompounds of formula (I) under mild operative is conditions, in highyields and purity.

In addition, it enables the preparation of the aforementioned compoundswithout the need of carrying out several steps and/or isolating manyintermediates which could lead to undesired by-products.

DETAILED DESCRIPTION OF THE INVENTION

Preferably, the process of the invention is directed to the preparationof the compound of formula (I) wherein R is a bromine atom, that is thecompound formerly indicated as PNU 166196. In this respect, it is clearto the man skilled in the art that within the general formula (I)compounds thus prepared, the possibility for the R group of beingbromine or chlorine will depend upon the compound of formula (V) beingused in step c).

According to the process object of the invention, the reaction of stepa) is carried out between 2-amino-ethylguanidine, optionally in the formof a pharmaceutically acceptable salt, preferably as2-amino-ethylguanidine dihydrochloride, and a slight excess, forinstance from 1 to 2 equivalents, of the compound of formula (II).

Within the compounds of formula (II) the X group represents hydroxy or asuitable leaving group such as, for instance, bromine, chlorine,2,4,5-trichlorophenoxy, 2,4-dinitrophenoxy, succinimido-N-oxy orimidazolyl.

Preferably, the X group is a bromine or chlorine atom.

The reaction is performed under basic conditions with from 1 to 4equivalents of an organic or inorganic base such as, for instance,sodium or potassium hydroxide, carbonate or bicarbonate, or with anorganic amine such as triethylamine, ethyldiisopropylamine, piperidineand the like.

Sodium carbonate or bicarbonate are preferably used. The reaction occursin the presence of a suitable solvent such as, for instance, dioxane,tetrahydrofuran, dimethylsulfoxide, dimethylformamide, water andadmixtures thereof.

According to a preferred embodiment of the process, step a) ispreferably carried out in the presence of dioxane, tetrahydrofuran,water or admixtures thereof.

The reaction temperature may vary from about 0° C. to about 50° C. andfor a time varying from about 1 to about 24 hours. The intermediatenitro-compound of formula (III) may be isolated, either as such or inthe form of a pharmaceutically acceptable salt, for instance ashydrochloride salt, or may be further processed, without being isolated,under reductive conditions as per step b) of the process.

The reduction of the compound of formula (III), as per step b) of theprocess, is carried out according to well-known methods for reducingnitro-derivatives to amino-derivatives.

Typical reductive conditions include the use of conventional reducingagents such as, for instance, sodium hypophosphite, hydrazine, sulfides,polysulfides and the like, or catalytic hydrogenation.

According to a preferred embodiment of the invention, the compound offormula (III) is hydrogenated under heterogeneous catalytic conditionsin the presence of platinum or palladium catalysts, for instancepalladium on charcoal (Pd/C).

The hydrogenation reaction is preferably carried out in the presence ofa suitable solvent, for instance dioxane, tetrahydrofuran,dimethylsulfoxide, dimethylformamide, water and admixtures thereof, attemperatures varying from about 0° C. to about 50° C., for a time ofabout 1 to about 24 hours and by employing from about 1 to about 10 bar(1 bar=10⁵ Pa) of hydrogen pressure.

The amino-derivative thus obtained is further reacted, without beingisolated, with the compound of formula (II). The reaction occurs underthe above reported conditions as per step a). of the process, that is tosay in the presence of a base and of a suitable solvent.

As formerly indicated, the intermediate nitro-compound of formula (IV)may be isolated, either as such or in the form of a pharmaceuticallyacceptable salt, for instance as hydrochloride salt, or may be furtherprocessed, without being isolated, in step c) of the process.

In step c), the reduction of the compound of formula (IV) is carried outaccording to well-known methods for reducing nitro-derivatives toamino-derivatives, as above reported. Preferably, the reaction iscarried out under catalytic hydrogenation conditions in the presence ofpalladium or platinum catalysts, as set forth above.

The resultant amino-derivative is then reacted, without being isolated,with a compound of formula (V) according to conventional methods for theacylation of amino derivatives.

In particular, the reaction is performed in the presence of a solventsuch as, for instance, dioxane, tetrahydrofuran, water,dimethylsulfoxide, dimethylformamide or admixtures thereof, in thepresence of a conventional condensing agent and of an inorganic ororganic base such as, for instance, sodium or potassium hydroxide,carbonate or bicarbonate, or with an organic amine such as, forinstance, triethylamine, ethyldiisopropylamine, piperidine and the like.

Preferred condensing agents are, for instance,N,N′-dicyclohexylcarbodiimide (DCC) or(N′-(3-dimethylaminopropyl)-N-ethylcarbodiimide) hydrochloride (EDC).

The reaction temperature may vary from about −10° C. to about 50° C. andfor a time varying from about 1 to about 24 hours. As formerlyindicated, the preparation of the compound of formula (I) wherein R is abromine atom (PNU 166196), as per step c) is carried out with a compoundof formula (V) wherein R is a bromine atom.

According to a preferred embodiment of the invention, the whole processcan be carried out in the presence of a unique reaction solvent, forinstance dioxane, tetrahydrofuran, water or admixtures thereof.

As such, it is clear that by carrying out the whole process in the samereaction solvent, volumes and, hence, large amounts of solvent to berecovered, are dramatically reduced.

According to a particularly preferred embodiment of the invention, allthe reaction steps from a) to c) can be carried out in one pot withoutthe need of isolating any intermediate.

In this respect, each of the reactions defined in steps from a) to c)are performed as follows, by first reacting a compound of formula (II)with 2-amino-ethylguanidine dihydrochloride so as to obtain a compoundof formula (III), by reducing the compound of formula (III) to thecorresponding amino-derivative and by subsequently reacting it with,again, the compound of formula (II) so as to obtain the compound offormula (IV) and, finally, by reducing it to the amino-derivative and byreacting this latter with the compound of formula (V).

Given the above, it is also clear to the man skilled in the art that,whenever desired, each, or at least some, of the aforementionedreactions can be alternatively accomplished by separating anyintermediate compound.

So far, any of the aforementioned approaches of the process forpreparing the compounds of formula (I) are within the scope of thepresent invention.

Finally, the conversion of the distamycin derivative of formula (I) intoa pharmaceutically acceptable salt or, on the other side, the conversionof a salt thereof into the free compound, may be carried out accordingto well known techniques.

Examples of pharmaceutically acceptable salts of the compounds offormula (I) are the acid addition salts with pharmaceutically acceptableacids such as, for instance, hydrochloric, hydrobromic, sulfuric,nitric, acetic, trifluoroacetic, propionic, succinic, malonic, citric,tartaric, methanesulfonic, p-toluensulfonic acid and the like.

The compound 2-ethyl-aminoguanidine, for instance as dihydrochloridesalt, is a known compound which can be prepared according to knownmethods; see, for instance, Syntethic Communications 20(16), 2559-2564(1990).

The compound of formula (II) is known or can be easily preparedaccording to known methods; for a reference to the preparation of thecompound of formula (II) wherein X is hydroxy see, as an example, U.S.Pat. No. 4,942,227.

The compound of formula (V), in its turn, is a commercially availablecompound.

From the above, it is worth noting that the process of the inventionallows to perform a set of subsequent reactions by using a limitednumber of reactive derivatives.

The compound of formula (II), in fact, is very conveniently used in twodifferent reactions of the process: first, as a starting material with2-aminoethylguanidine dihydrochloride, in step a), and subsequently as areactive intermediate in step b).

The intermediate compound of formula (IV) is novel and, hence,represents a further object of this invention. As formerly indicated,the compounds of formula (I) are useful in therapy as antitumor agents.

For a general reference to the antitumor activity of the compounds offormula (I) see the aforementioned WO 98/04524.

According to a practical embodiment of the process of the invention forpreparing the compound of formula (I) wherein R is a bromine atom (PNU166196), a proper amount of 2-amino-ethylguanidine dihydrochloride isreacted, in a solvent system preferably comprising dioxane,tetrahydrofuran, water or admixtures thereof and in the presence of abase, for instance an inorganic base such as sodium carbonate orbicarbonate, with a proper amount, preferably a slight excess, of thecompound of formula (II).

The reaction is carried out under mild operative conditions and theresultant nitro-derivative of formula (III) is first hydrogenated underheterogeneous catalytic conditions, in the presence of palladium oncharcoal, and subsequently reacted with a proper amount, preferably aslight excess, of the compound of formula (II), under basic conditions.

The resultant compound of formula (IV) is then hydrogenated as abovedescribed to the corresponding amino-derivative which is furtherreacted, in the presence of a base and of a condensing agent, forinstance EDC, with α-bromoacrylic acid of formula (V).

The desired compound of formula (I) thus obtained is then isolated inhigh yields and purity, according to conventional methods.

With the aim of illustrating the present invention, without posing anylimitation to it, the following examples are now given.

EXAMPLE 1

Preparation of Tert-butyl-N-(2-aminoethyl)carbamate

Ethylendiamine (7 moles) was loaded into a reaction flask containingdioxane (2.7 l). Di-tert-butyl-dicarbonate (1.0 mole) in dioxane (270ml) was subsequently added.

The reaction mixture was stirred at room temperature for 1 day and thesolvent was then removed under vacuum.

Water (1.8 l) was added to the crude and the mixture was extracted withdichloromethane. The organic solvent was distilled off from the organicphase thus yielding the title compound (145 g; 90% yield).

EXAMPLE 2

Preparation of 2-aminoethyl-guanidine dihydrochloride

Tert-butyl-N-(2-aminoethyl)carbamate (0.4 moles), O-methylisoureahydrogenosulphate (0.8 moles) and triethylamine (2.5 moles) were loadedinto a reaction flask containing a methanol:water=1:1 mixture (3 l).

The reaction mixture was stirred at room temperature for one day and thesolvent was then removed under vacuum.

The crude was treated with ethanol (2 l) and gaseous hydrochloric acid(3.5 M) at 20° C. for 4 hours and subsequently filtered and dried, thusyielding the title compound (55 g; 74% yields).

EXAMPLE 3

Preparation of N-methyl-4-aminopyrrole-2-carboxylic acid

N-methyl-4-nitropyrrole-2-carboxylic acid (0.58 moles), hydrochloricacid 2N (350 ml) and Pd/C (5 g) were added to a dioxane:water=2:1mixture (1.2 l).

The mixture was hydrogenated into a 2 l hydrogenation reactor at roomtemperature for 4 hours. The catalyst was filtered off, the solventconcentrated under vacuum and the resultant suspension was filtered.

The crude was dried yielding the title compound (94 g; 92% yield) as awhite powder.

EXAMPLE 4

Preparation of N-methyl-4-nitropyrrole-2-carboxyxlic acid chloride

Thionyl chloride (3.16 moles) was loaded into a reaction flaskcontaining N-methyl-4-nitropyrrole-2-carboxylic acid (1.17 moles) intoluene (1.5 l).

The reaction mixture was stirred for 3 hours at 100° C., cooled andsubsequently concentrated under vacuum.

The resulting suspension was treated with cyclohexane for 2 hours atroom temperature, then filtered and dried yielding the title compound(200 g; 96% yield).

EXAMPLE 5

Preparation of N-methyl-4-[(N′-methyl-4-nitro-pyrrolyl-2yl)carbonylamino]pyrrole-2-carboxylic acid

N-methyl-4-aminopyrrole-2-carboxylic acid (0.56 mole), prepared asdescribed in example 3, and sodium bicarbonate (2 moles) were loadedinto a reaction flask containing a dioxane:water=1:1 admixture (300 ml).

A solution of N-methyl-4-nitropyrrole-2-carboxylic acid chloride (0.62moles), prepared as described in example 4, in dioxane (350 ml) was thenadded therein.

The reaction mixture was stirred for 1 hour at room temperature, thenwater (100 ml) was added.

A solution 2N of hydrochloric acid was then added up to pH 3 and theorganic solution was concentrated under vacuum until dioxane wascompleted removed

The suspension was filtered yielding the title compound (147 g; 90%yield).

EXAMPLE 6

Preparation ofN-methyl-4-[(N′-methyl-4-nitro-pyrrolyl-2-yl)carbonylamino]pyrrole-2-carboxylicacid chloride

N-methyl-4-[(N′-methyl-4-nitro-pyrrolyl-2-yl)carbonylamino]pyrrole-2-carboxylicacid (0.45 moles), prepared as described in example 5, dimethylformamide(3 ml) and thionyl chloride (2.3 moles) were loaded into a reactionflask containing dichloromethane (2.5 l).

The resultant suspension was stirred for 6 hours at refluxingtemperature, then cooled at room temperature and filtered.

The crude was treated with hexane (1 l), then filtered and driedyielding the title compound (163 g; 90% yield).

EXAMPLE 7

Preparation ofN-methyl-4-[(N′-methyl-4-nitro-pyrrolyl-2-yl)carbonylamino]pyrrole-2-carboxylicacid chloride ethylguanidine hydrochloride.

N-methyl-4-[(N′-methyl-4-nitro-pyrrolyl-2-yl)carbonylamino]pyrrole-2-carboxylicacid chloride (0.057 moles), prepared as described in example 6, wasloaded into a reaction flask containing dioxane (300 ml).

Then, a solution of 2-aminoethyl-guanidine dihydrochloride (0.057moles), prepared as described in example 2, and sodium bicarbonate (0.17moles) in water (100 ml) were added therein.

The suspension was stirred for 2 hours at room temperature. A solution2N of hydrochloric acid (60 ml) and Pd/C (3 g) were subsequently added.

The mixture was then hydrogenated into a 2 l hydrogenation reactor atroom temperature for 3 hours and then filtered.

The resultant solution was then added to a suspension ofN-methyl-4-[(N′-methyl-4-nitro-pyrrolyl-2-yl)carbonylamino]pyrrole-2-carboxylicacid chloride (0.057 moles) in dioxane (120 ml). Sodium bicarbonate(0.17 moles) was then added and the suspension was stirred at roomtemperature for 4 hours.

The mixture was then concentrated under vacuum until a suspension wasobtained and the resulting suspension was cooled at room temperature andfiltered.

The wet crude was treated with acetone, filtered and dried thus yieldingthe title compound (30 g; 85% yield).

EXAMPLE 8

Preparation of2-[1-methyl-4-[1-methyl-4-[1-methyl-4-(1-methyl-aminopyrrole-2-carboxamido)pyrrole-2-carboxamido]pyrrole-2-carboxamido]pyrrole-2-carboxamido]ethylguanidinehydrochloride

The compound of example 7 (0.067 moles), dioxane (550 ml), water (450ml), a solution 2N of hydrochloric acid (120 ml) and Pd/C (13 g) wereloaded into a hydrogenation reactor.

The suspension was hydrogenated at room temperature for 3 hours, thenthe mixture was filtered.

The resultant solution was treated with acetone (1300 ml), cooledovernight at 4° C. and filtered.

The crude was dried thus yielding the title compound (40 g; 90% yield.

EXAMPLE 9

Preparation ofN-(5-{[(5-{[(5-{[(2-{[amino(imino)methyl]amino}ethyl)amino]carbonyl}-1-methyl-1H-pyrrol-3-yl)amino]carbonyl}-1-methyl-1H-pyrrol-3-yl)amino]carbonyl}-1-methyl-1H-pyrrol-3-yl)-4-[(2-bromoacryloyl)amino]carbonyl-1-methyl-1H-pyrrole-2-carboxamidehydrochloride (internal code PNU 166196A).

Bromoacrylic acid (7.56 mmoles), EDC (7.56 mmoles), sodium bicarbonate(14.3 mmoles) and dioxane (60 ml) were loaded into a reaction flask.

The compound of example 8 (2 mmoles) and a mixture of dioxane:water 2:1(30 ml) were then added. The mixture was stirred for 1 hour at roomtemperature and a solution 2 N of hydrochloric acid was then added up topH 4.5.

The solvent was then concentrated under vacuum, the suspension wasfiltered and the resultant crude was dried yielding the title compound(1.3 g; 85% yield; HPLC Area>98%).

1. A process for preparing a distamycin derivative of formula

wherein R is a bromine or chlorine atom; or a pharmaceuticallyacceptable salt thereof; which process comprises: a) reacting, underbasic conditions, 2-amino-ethylguanidine with a compound of formula

 wherein X is hydroxy or a suitable leaving group, so as to obtain acompound of formula

b) reducing the nitro-derivative of formula (III) to the correspondingamino-derivative and, subsequently, reacting the resultantamino-derivative with the above compound of formula (II), under basicconditions, so as to obtain a compound of formula

c) reducing the nitro-derivative of formula (IV) to the correspondingamino-derivative and, subsequently, reacting in the presence of asuitable condensing agent and under basic conditions the resultantamino-derivative with a compound of formula

 wherein R is a bromine or chlorine atom; so as to obtain the compoundof formula (I) and, optionally, converting it into a pharmaceuticallyacceptable salt thereof.
 2. A process according to claim 1 for thepreparation of the compound of formula (I), or a pharmaceuticallyacceptable salt thereof, wherein R is a bromine atom.
 3. A processaccording to claim 1 wherein steps a) to c) are carried out in one potwithout isolating any reaction intermediate.
 4. A process according toclaim 1 wherein, in step a), 2-amino-ethylguanidine is in the form ofits dihydrochloride salt.
 5. A process according to claim 1 wherein,within the compound of formula (II) of step a), X is hydroxy or a groupselected from bromine, chlorine, 2,4,5-trichlorophenoxy,2,4-dinitrophenoxy, succinimido-N-oxy or imidazolyl.
 6. A processaccording to claim 5 wherein X is a bromine or chlorine atom.
 7. Aprocess according to claim 1 wherein the reductive steps under b) or c)are carried out in the presence of a reducing agent selected from thegroup consisting of sodium hypophosphite, hydrazine, sulfides, andpolysulfides or as catalytic hydrogenation reactions.
 8. A processaccording to claim 7 wherein the reductive steps b) or c) are carriedout as catalytic hydrogenation reactions in the presence of palladium orplatinum hydrogenation catalysts.
 9. A process according to claim 1wherein, in step c), the reaction between the resultant amino derivativeand the compound of formula (V) is carried out in the presence of asuitable condensing agent selected from the group consisting ofN,N′-dicyclohexylcarbodiimide (DCC) or(N′-(3-dimethylaminopropyl)-N-ethylcarbodiimide) hydrochloride (EDC).10. A process according to claim 1 wherein any reaction performed underbasic conditions is carried out in the presence of an inorganic ororganic base.
 11. A process according to claim 10 wherein the base issodium or potassium hydroxide, carbonate or bicarbonate, triethylamine,ethyldiisopropylamine or piperidine.
 12. A process according to claim 1wherein the reactions of steps a) to c) are, each independently, carriedout in the presence of a suitable reaction solvent selected from thegroup consisting of dioxane, tetrahydrofuran, dimethylsulfoxide,dimethylformamide, water or admixtures thereof.
 13. A process accordingto claim 12 wherein the reaction solvent is selected from the groupconsisting of dioxane, tetrahydrofuran, water and admixtures thereof.14. A process according to claim 1 wherein said pharmaceuticallyacceptable salts of the compounds of formula (I) are the acid additionsalts of pharmaceutically acceptable acids selected from the groupconsisting of hydrochloric, hydrobromic, sulfuric, nitric, acetic,trifluoroacetic, propionic, succinic, malonic, citric, tartaric,methanesulfonic and p-toluensulfonic acid. 15.